First report of barley root-knot nematode, Meloidogyne naasi from turfgrass in Idaho, with multigene molecular characterization

Abstract Barley root-knot nematode, Meloidogyne naasi Franklin, 1965, is one of the most important pest nematodes infecting monocots (Franklin, 1965). Two-inch core soil samples collected from a golf course in Ada County, Idaho were submitted for identification in November of 2019. A high number of Meloidogyne sp. juveniles were recovered from both soil samples using sieving and decantation followed by the sugar centrifugal flotation method. They were examined by light microscopy, morphometric measurements, and multiple molecular markers, including the ribosomal 28S D2–D3 and intergenic spacer 2 (IGS-2) regions, mitochondrial markers cytochrome oxidase I (COI) and the interval from COII to 16S, and the protein-coding gene Hsp90. Morphometrics as well as BlastN comparisons with other root-knot nematode sequences from GenBank were consistent with identification as M. naasi. Phylogenetic trees inferred from 28S, IGS-2, COI, or Hsp90 alignments each separated the Idaho population into a strongly supported clade with other populations of M. naasi, while the COII-16S interval could not resolve M. naasi from M. minor. This report represents the first morphological and molecular characterization of Meloidogyne naasi from turfgrass in Idaho.

Turfgrasses are widely used as groundcovers in a variety of sporting, residential, and recreational contexts, and maintaining high quality and aesthetics is an essential feature of turfgrass management.Control of newly emerging diseases and nematodes presents an ongoing challenge to the health, quality, production, and maintenance of turfgrass.The barley root-knot nematode, Meloidogyne naasi Franklin, 1965, was originally described in field crops (cereals, grasses, and sugarbeet, Beta vulgaris L.) in England and Wales (Franklin, 1965).This species is one of the most important root-knot nematodes (RKN) affecting monocots, and is known to cause reduced growth and vigor in turfgrasses (Bélair et.al., 2006).This species has a wide distribution, having been reported in the United States in several states, as well as in Canada, South America and Europe (Zeng et al., 2012).A report from 2012, in which authors surveyed 238 golf courses from 10 states across the western United States, found RKN in 60% of the samples (McClure et.al. 2012).Molecular analysis of specimens from 110 golf courses revealed that the most prevalent root-knot nematode species was M. naasi, which was found in 58 samples (McClure et al., 2012).
A similar study by Ye et al. (2015) analyzed data from 51 RKN populations collected from turfgrasses within the eastern United States.In contrast with the western U.S. golf courses surveyed, the most prevalent RKN species on the East Coast were M. marylandi and M. graminis (McClure et al., 2012;Ye et al., 2015).Meloidogyne naasi was also identified, but was not present in as many samples as M. marylandi and M. graminis (Ye et al., 2015).More recently, Meloidogyne naasi was found on a football field in Porto, Portugal (Vieira dos Santos et al., 2020).
The Banbury Golf Course in Bandon, Oregon reported a poorly performing section that had been planted entirely from seed, including a majority percentage of Poa annua (via overseeding the original bentgrass/fescue seed mix) (Fig. 1).Preliminary analysis by Rutgers University identified Anguina pacificae in these samples, but subsequent examination by diagnosticians at Oregon State University and our laboratory failed to repeat that finding.From the two soil samples received for identification, only juveniles were recovered.No mature females or males were found.The morphometric details of the juveniles were recorded and compared to the original description of M. naasi (Franklin, 1965) and others that followed (Karssen et al., 2002;Zhao et al., 2017, Suresh et al., 2017and Santos et al., 2020).Sequences from the large subunit ribosomal DNA (28S), intergenic region (IGS-2), the mitochondrial DNA interval between cytochrome oxidase II (COII) and the 16S gene, partial mitochondrial cytochrome oxidase I (COI), and nuclear heat shock protein (Hsp90) gene were obtained and compared to the existing information in

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GenBank.Based upon the unambiguous similarity of these DNA markers with those previously reported for the species (McClure et al., 2012;Nischwitz et al., 2013) and the morphological data, we identify this isolate as Meloidogyne naasi.Prior evidence of M. naasi in Idaho was reported by Powers et al. (2018), who recovered a COI sequence from RKN on potato.This report represents the first morphological and molecular description of Meloidogyne naasi from Idaho turfgrass.

Materials and Methods
Two soil samples were sent to the USDA Mycology and Nematology Genetic Diversity and Biology Laboratory (Beltsville, MD) in November of 2019.The origin of the soil samples was Banbury Golf Course in Ada County, Idaho.Nematodes were recovered from soil using nested 20, 60, and 500 µm sieves, with juveniles (J2s) recovered from the 500 µm sieves using the sieving decanting method, followed by sugar centrifugal flotation (Jenkins, 1964).Nematodes were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Golden, 1990;Hooper, 1970).Females recovered from the soil samples were fixed in 3% formaldehyde solution.Photomicrographs of the specimens were made with a Nikon Eclipse Ni compound microscope, using a Nikon DS-Ri2 camera (Nikon USA, Melville, NY).Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope (Leica, Wetzlar, Germany).All measurements are in micrometers unless otherwise stated.
Other PCR amplicons were sequenced directly with their corresponding PCR primers, except the 28S which was sequenced with the primers D3A and D3B.All PCR and cloned products were sequenced by Genewiz, Inc. (South Plainfield, NJ, USA).GenBank accession numbers were assigned as follows: 28S rDNA (MT406252); IGS-2 rDNA (OQ72994 to OQ72997); mitochondrial COII to 16S DNA (MT408951 to MT408953); COI (MT408951 to MT408953); and Hsp90 (MT408947 to MT4089450).Newly obtained sequences were compared with publicly available sequences in GenBank using BlastN.Multiple sequence alignments of each marker were constructed with ClustalW or MAFFT within Geneious, with outgroups chosen based on previous studies.Phylogenetic trees were constructed by Bayesian Inference (BI) under the GTR + I + G model implemented within the MrBayes plugin in Geneious.BI analysis for each gene included a random starting tree and was run with four chains for 2 × 10 6 generations.Two runs were performed for each analysis.Trees were sampled every 1000 th generation, with 25% of results discarded as burnin.Remaining samples were used to generate a 50% majority-rule consensus tree.Posterior probabilities (PP) are shown on appropriate clades.

Results and Discussion
Morphological Analysis: Morphological measurements of M. naasi juveniles (n = 30) included body length, maximum body width, stylet length, body width at anus, tail length, tail terminus length, and a and c values (Table 1).Four lines were noted in the lateral field.All measurements were consistent with values previously established for M. naasi 6.4 ± 0.3 (6.0 -6.7) 6.4 ± 0.8 (5.6 -8.0) *Values were calculated based on 10 specimens.
JOURNAL OF NEMATOLOGY (Franklin, 1965;Karssen et al., 2002;Zhao et al., 2017, Suresh et al., 2017and Santos et al., 2020).Images from second-stage juveniles are shown in Fig. Amplification of the IGS-2 rDNA from three J2 gave two identical sequences and a third that was 99.6% identical (7 bp differences).These IGS-2 sequences from M. naasi are the first to be recovered from this species, and no others were available in GenBank for comparison.Four additional populations identified as M. naasi in a previous study from golf course turfgrass were also subjected to PCR of IGS-2 (McClure et al., 2012).These sequences ranged from 99.3 to 100% identical (0 to 9 bp differences) to the Idaho population.The other highest similarity matches included M. oryzae (LS974439), with 73% identity.
The phylogenetic tree (Fig. 4) inferred from alignment of newly obtained M. naasi IGS-2 and other RKN sequences placed the Idaho population in a clade along with two M. naasi isolates from California and one from Washington with maximum support (PP = 1.0).The M. naasi sequences were grouped near M. minor and a clade containing M. fallax and M. chitwoodi.Other IGS-2 sequences from species included in the 28S tree were not available for further comparison of their topologies.
PCR of the mitochondrial interval COII-16S from three J2 gave two identical sequences, and a third that varied at 3 bp from the others (99.6 to 100% identity).BlastN yielded top hits to both M. naasi and M. minor sequences (between 0 and 7 bp differences, with 99.1 to 100% identity).The Bayesian tree constructed from a multiple sequence alignment of selected Meloidogyne populations (Fig. 5) showed that M. naasi and M. minor formed a polyphyletic clade, and that COII-16S could not reliably resolve these species.In a previous study of RKN populations from golf course turfgrass (McClure et al., 2012), mtCOII-16S interval could not distinguish populations M. fallax and M. chitwoodi, with sequences from several geographically diverse populations differing at only a single deletion or insertion.Thus, this locus is not able to resolve some RKN species.
The presence of mixed M. naasi and M. minor populations on golf-course turfgrass has created challenges for molecular species discrimination, and care must be taken to ensure that specimens in a mixture are carefully separated before molecular analysis.Meloidogyne minor is a relatively new species first found on potato in the Netherlands in 2004, on sports fields in the United Kingdom (Karssen et al., 2004), and in Belgium, Chile, and New Zealand (Zhao et al., 2017).Unfortunately, mtCOII-16S sequences from the latter countries were unavailable, precluding a broader geographic comparison of diversity in this marker.
Amplification of mitochondrial COI gave two identical sequences from separate J2.BlastN comparison to GenBank resulted in matches to M. naasi sequences KU517170 (origin not given) and MH128479 (reported from potato in Idaho; Powers et al., 2018) at between 99.4 and 99.5% identities (2 to 3 bp differences).Additional M. naasi matches included longer COI sequences (763 bp) generated with different primers (Powers et al., 2018) that varied at 20 to 23 bp over a 397-bp region in common.Sequences from M. naasi (KM491208, KM49121 to KM49125) only overlapped at 105 bp with the fragments generated here, with those matching 100%.
In the Bayesian tree resulting from the multiple sequence alignment generated with selected RKN species (Fig. 6), all M. naasi sequences formed a clade with maximum PP = 1.00.Sequences from M. chitwoodi and M. fallax grouped together, separated by 5 bp, as shown in a prior study (Powers et al., 2018).
Hsp90 amplification from three juveniles resulted in four cloned PCR products varying in length from 1031 to 1042 bp, with 98.8 to 99.4% identity (6 to 12 bp differences) among clones.BlastN comparison to sequences in GenBank indicated that the best matches to M. naasi were previously identified from golf courses in the western U.S. (KC262248 to KC262252) with 99.0 to 99.9% identity (1 to 9 bp differences).The next closest similarity was to

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Based upon the unambiguous similarity of these DNA markers with those previously reported for the species (McClure et al., 2012, Nischwitz et al., 2013, Hodgetts et al., 2016;Powers et al., 2018) and the morphological data (Franklin, 1965;Karssen et al., 2002;Zhao et al., 2017, Suresh et al., 2017;Santos et al., 2020), we identify this isolate as Meloidogyne naasi.For each marker examined, the phylogenetic groupings of species were consistent, limited only by the availability of sequences from each species, with M. naasi appearing in Clade III, as defined M. minor (KC262245) and several others, at 85.47% similarity (145 bp differences).In the Bayesian tree inferred from alignment of selected RKN Hsp90 sequences (Fig. 7), M. naasi formed a maximally supported clade.Other distinct clades with maximum support values were obtained for M. minor, M. fallax, M. chitwoodi, and an unnamed RKN species isolated from garlic mustard.Tropical species M. floridensis, M. incognita, M. arenaria, and M. javanica could not be reliably separated by Hsp90, although M. enterolobii grouped separately from those species.previously in a 5-gene phylogeny by Alvarez-Ortega et al. (2019).This clade includes nine species from monocots and dicots that reproduce exclusively by meiotic parthenogenesis.Except for M. salasi, found only in South America, and M. kralli, found only in Europe, the rest of the species in Clade III (M. trifoliophila, M. graminicola, M. naasi, M. exigua, M.chitwoodi, M. fallax, and M. minor) are found on multiple continents.
The greens at Banbury Golf Course were originally planted as creeping bent grass (Agrostis stolonifera 'Dominant Plus').The course originated in 1999.The greens are currently a mixture of creeping bent grass and annual bluegrass (Poa annua).Nematode damage has been observed in the spring, summer, and fall.The patches often reappeared in identical or very similar locations.Most of the greens (20 total, including the practice putting green and the chipping green) have seen damage.A few show no signs of nematodes.Interestingly, the damage often impacts the bent grass and not the annual bluegrass (Darryl Glinski, personal communication).
This study represents the first report of M. naasi from turfgrass in Idaho, accompanied by micrographs, morphometrics, and multi-gene characterization.This adds to a previous study by Powers et al. (2018), who The nematodes in the present work were isolated from soil samples taken from golf course greens, not from the roots themselves, although the turf was reported by the golf superintendent to be in decline.Interestingly, no other species of RKN were found in the samples.Mixtures of RKN species on golf course greens were previously reported by McClure et al. (2012), and indeed, some of the samples included in that study required careful microscopic examination of J2 to manually separate M. naasi from M. minor.
In the United Kingdom, mixed populations of M. naasi and M. minor are common.Meloidogyne naasi was the most prevalent RKN detected in a survey of 11 golf courses and eight football pitches in Belgium (Vandenbossche et al., 2011).In Washington State, these species were detected together on golf course greens in ratios of approximately 4:1.In a study of nematodes identified by diagnostic laboratories in the Pacific Northwest (Zasada et al., 2019), M. naasi was found in fewer than 1% of samples, making them much less common than M. chitwoodi (found in 71%

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Supplementary Table S1.Root knot nematode isolates and GenBank numbers included in this study.

First
report of Meloidogyne naasi from Idaho: Skantar et al.

Figure 1 :
Figure 1: Damage on golf course turfgrass in Banbury, Idaho. A. Golf course green in decline, showing patchy surface.B. Close-up of damage to mixed turfgrass, associated with M. naasi.Photo credits: Darryl Glinski.
2. Molecular Analysis: Amplification of the IGS-2 ribosomal DNA region and the D2-D3 expansion segment of region 28S yielded sequences of 1334 to 1340 bp and 775 bp, respectively.Amplification of the mitochondrial interval between COII and the large (16S) rRNA gene yielded sequences of 416 to 441 bp, and from the COI gene, 752 to 796 bp.Hsp90 PCR generated sequences ranging from 1031 to 1043 bp.Amplification of the D2-D3 expansion region of 28S rDNA yielded identical sequences from three individual J2.Similarity matches from BlastN against the GenBank database included other M. naasi sequences at 100%, with full coverage.These matches included accessions KP901068 and KP901069 representing populations obtained from bentgrass in NC, and several others from the U.S. (McClure et al., 2012) and Portugal (Vieira dos Santos et al., 2020), with > 99% similarity (2 to 9 bp differences).Bayesian phylogenetic trees constructed from multiple sequence alignments of Meloidogyne spp.placed the Idaho population in a clade with other M. naasi sequences at PP = 1.00 support Fig. 3.This clade was in a sister group with M. trifoliophila, M. graminicola and M. salasi with maximal support.

First
report of Meloidogyne naasi from Idaho: Skantar et al.

Figure 3 :
Figure 3: Phylogenetic relationships of Idaho population of Meloidogyne naasi and other selected root-knot nematodes, as inferred from a 735-bp alignment of 28S rDNA sequences, with M. artiellia as the outgroup.A 50% majority rule consensus tree obtained from Bayesian analysis was generated using the GTR + I + G model of nucleotide substitution.Branch support (PP) values above 0.5 are shown on appropriate branches.New sequences are highlighted in blue bold font.

Figure 4 :
Figure 4: Phylogenetic relationships of Idaho population of Meloidogyne naasi and other selected root-knot nematodes, as inferred from a 1764-bp alignment of IGS-2 rDNA sequences, with M. hapla as the outgroup.A 50% majority rule consensus tree obtained from Bayesian analysis was generated using the GTR + I + G model of nucleotide substitution.Branch support (PP) values above 0.5 are shown on appropriate branches.New sequences are highlighted in blue bold font.

First
report of Meloidogyne naasi from Idaho: Skantar et al.

Figure 5 :
Figure 5: Phylogenetic relationships of Idaho population of Meloidogyne naasi and other selected root-knot nematodes, as inferred from a 1764-bp alignment of mitochondrial sequences including the interval from COII to 16S, with M. hapla as the outgroup.A 50% majority rule consensus tree obtained from Bayesian analysis was generated using the GTR + I + G model of nucleotide substitution.Branch support (PP) values above 0.5 are shown on appropriate branches.New sequences are highlighted in bold font; other M. naasi is in blue and M. minor in red font.

Figure 6 :
Figure 6: Phylogenetic relationships of Idaho population of Meloidogyne naasi and other selected root-knot nematodes, as inferred from a 763-bp alignment of mitochondrial COI sequences, with M. artiellia as the outgroup.A 50% majority rule consensus tree obtained from Bayesian analysis was generated using the GTR + I + G model of nucleotide substitution.Branch support (PP) values above 0.5 are shown on appropriate branches.New sequences are highlighted in blue bold font.The COI sequence from Powers et al. (2018) is marked by an asterisk.

First
report of Meloidogyne naasi from Idaho: Skantar et al.

Figure 7 :
Figure 7: Phylogenetic relationships of Idaho population of Meloidogyne naasi and other selected root-knot nematodes, as inferred from a 1003-bp alignment of Hsp90 genomic sequences, with M. hapla as the outgroup.A 50% majority rule consensus tree obtained from Bayesian analysis was generated using the GTR + I + G model of nucleotide substitution.Branch support (PP) values above 0.5 are shown on appropriate branches.New sequences are highlighted in blue bold font.

Table 1 :
Morphometrics of infective second-stage juveniles of Meloidogyne naasi.Values expressed in the form mean +/-standard deviation (range).